Flash Study Flashcards
Termination of transcription
1) mGC rich stretch followed by a polyA on the template strand. The GC in the RNA pair forming a dsRNA (hairpin loop). polyU
2) rho-dependent termination: Rho binds to the rutsite in RNA. After binding, pulls RNA off the RNA polymerase. rut is close to sequences at which RNA polymerase pauses.
Footprinting 5
1) A sequence of DNA bound to a protein (RNA polymerase) is partially digested with DNase.
2) Cleaved positions are recognized by using DNA labeled in one strand at one end.
3) Broken DNA fragments are electrophoresed on a gel separating according to length of fragments.
4) In free DNA, every bond position is broken. But the region covered by RNA polymerase (protein) is protected.
5) Two reaction are run: free DNA (control) + a DNA-protein
Three major modifications of transcripts in eukaryotes
Processing:
•Methyl guanosine caps (MG caps) are added to the 5’ end.
•Non-coding regions (introns) are spliced out.
•Poly(A) tails are added to the 3’ (mediated by endonuclease and poly A polyeramse)
All of this phosphorylation of the DTD of RNApol is critical in signalling recruitment of these proteins.
Why Introns 3
- Important in gene regulation (rate of transcription)
- Evolutionary relics from gene-gene fusions
- alternative splicing (1 gene—>many products)
Lessons from RNAseq Stidies 3
1) New gene annotation (“gene discovery”)
2) Differential expression profiles between conditions. (miRNA cancer [only a small portion of tumor miRNAsare released into serum. They can be used as biomarkers], over expressed proteins in sterile males)
3) What is the nature of the regulatory difference?cis vs. trans regulation
the Interactome
yeast two hybrid cell systems —>prot-prot interactions. Only if protein X and Y interact, then the binding and activation domains come together to turn on a reporter gene. (Alzheimer’s disease: βactin [ACTB] 10x higher expression in AD cerebral cortex than controls. Abnormalities in actin cytoskeleton may promote synaptic failure.)
Protein DNA Interactions
CHIP (Chromatin Immunoprecipitation assays). Does a protein bind DNA under certain conditions? Treat cells grown under certain condition (e.g. high temperature) with a chemical that cross. Break chromatin and use antibodies as bait for your suspected proteins
How to deal with polygenic disease associations
SNP 1) focus on human populations with high instances of the disease (example P2) 2)map first region of the genome where major gene variants causing diseases reside 3)do large scale genome sequencing to find rare variants
Catabolic repression
- A catabolic product of glucose prevents induction by lactose catabolite repression
- When glucose has a high concentration, the cAMP concentration will be low.
- Catabolite repression is mediated by a catabolite activator protein (CAP) and cAMP. High glu inhibits adenylate cyclase –>low cAMP.
- The lac promoter has a CAP-cAMP binding site.
- Binding of CAP-cAMP to the promoter is needed for lac operon induction (positive control
- CAP (catabolite activator protein) made by the gene known as crp.
- Of course, mutants that are anything other than the normal lac operon, cannot convert ATP into cAMP and thus cannot produce β-galactosidase because of cAMP concentration not big enough.
How to measure transcription levels? 3
qRTPCR
gel assay
genome wide approach with CHIp but need antibody
Transcription regulatory proteins
- DNA binding,
- prot-prot interactions,
- sites for signals (eg. GR),
- Some can cause chromatin change
tryptophan operon
high tryptophan binds (allosteric site) activates a repressor. low tryptophan —>repressor inactive —>transcriptopn of enzymes needed to make tryptophan. Attenuation: If high tryp —> rapid translation—>termination of transcription—>no more Trp. if low Trp, translation stalls—>transcription continues
Lambda phage
Lysogeny: CI —>prepressor protein—>blocks promoters for transcription og lytic pathway genes .
Lytic: CI—>RNApol—>Cro—>blocks promoter for CI transcription + antiterminaotr proteins allow progression of transcription.
Under poor conditions—> Ciii—>Cii Cii—>recuit polymerase for: Cro amtisease; CI sense—>lysongeny.
Good conditions —>CIII—>CII is degrated—>RNApol transits Cro—>lysis
Yeast GAL system
In the presence of galactose, GAL4 protein (trans element) is produced which activates transcription of other galgenes coding for enzymes by binding upstream enhancers(UAS). A key regulator is Gal4. Regulates expression of Gal1/2/7/10. Each of these genes has 2+ 17bp Gal4 binding sites (enchancers). Two critical domains, the DNA-bidning domain and the activation domain are needed for Gal4 to funciton. Gal80 normally blocks Gal4 function unless Gal3 inactivates it. Gal 3 inly binds to Gal80 when it is bound to galactose. Gal4 can both attract other proteins involved in initiating transcription and recruit proteins to modify chromatin structure.
